Molecular Bacterial Load Assay as TB Treatment Response Marker

1. The 7th East Africa Health and Scientific ConferenceMolecular Bacterial Load Assay evaluated as a marker for treatment response late during treatment Ntinginya N,Sabiiti W, Kuchaka D, Phillips P, Kibiki G, Gillespie SH, Mtafya B, Hoelscher M, Heinrich N Dar Es Salaam 27-29 March 2019 National Institute for Medical Research www.nimr.or.tz

2. Background: Rationale 7m ? 6m ? 4m ? Effective methods to measure the success of TB treatment are urgently needed Why: benefit of individualized treatment • 79 - 89% patients cured within 4 months – shorter treatment possibleFox et al 1999, Gillespie et al 2014, Merle et al., 2014, Jindani et al., 2014 • 2.2 -3 % relapse after 6 months – benefit from extensionAung K et al, 2012

3. Background: Rationale Pre-clinical & Phase I Phase IIA 1 Mio € Phase IIB 1,5 - 2 Mio € 30 - 80 Mio € Phase III Registration Why: Better end points for TB drug trials needed • No missing data due to culture contamination • Time and cost saving Phillips et al 2015

4. Background: Current tools Mitchison et al.1993, Wallis et al., 2015, Phillips et al., 2016 Clinical and Chest Radiography: Unspecific Smear microscopy: • Low sensitivity WHO 2009, Aung et al., 2012ISC 3rd Edition 2014 Culture: Accepted marker in trial at month 2 not individual patient Biosafety concerns , long time to results, contamination, non-culturable bacilli

5. Background: Molecular tools • Poor marker for monitoring TB treatment response Friedrich S and Rachow A et al, 2013 DNA based assay (Xpert MTB/RIF) • Cannot differentiate dead or live bacilli

6. Background: Molecular tools RNA is cleared rapidly after initiation of TB therapy Molecular Bacterial Load Assay (MBLA); • Specific to Mtb (qPCR Mtb 16s rRNA) • Not affected by contamination • Uses standard curve to quantify bacterial load Heller et al., 1999, Honeyborne et al., 2011 & 2014 , Sabiiti 2017 (submitted)

7. Objective Methodology • Nested inPanACEA MAMS TB 01 study (MAMS) and PANBIOME. A digital platform was used to collect metadata (demographics &clinical) • details published by Boeree et al., 2016 Lancet ID • Culture and MBLA Source: Google map • To evaluate a Molecular Bacterial Load (MBL) Assay as a marker for monitoring treatment response among TB patients treated with different drug combinations

8. Results : MBLA and MGIT Comparison between MBLA and MGIT media: qualitative results Contamination is a REAL problem in liquid culture 830/2192 (38%) Pearson chi square P < 0.001

9. Results : MGIT- Qualitative Qualitative liquid MGIT media final results per visit from baseline to the end of treatment.

10. Results : MBLA- Qualitative Qualitative MBLA final results from baseline to the end of treatment

11. Results : MBLA-Qualitative Malherbe et al., 2016 reported MTB mRNA in 35% of culture-negative sputum samples at Month 6 Percent of positive tests of all tests with valid results over treatment

12. Conclusion RNA based assay (MBLA) bears potential; • Is more often positive than culture • No missing data due to contamination • No assay inhibition • Allows longer and consistent follow up profiles • Its use as a study endpoints

13. Limitations Our study on MBLA in this work had several limitations • Only compared to culture, an imperfect marker for outcome • Insufficient data on long term clinical outcome • Different drug regimen with differing mode of action

14. Outlook Thank you for the accorded attention Moving forward with the MBLA • Prognostic value using clinical long term outcome • Implementation study in routine patients • Further validation as endpoint in TB drug trials

15. Acknowledgement CIH Coordination • University of St. Andrews (Stephen Gillespie and Wilber Sabiit) • Study participants • Dr. Norbert Heinrich,, Dr. Leonard Maboko and Prof Dr. Michael Hoelscher • Dr. Andrea Rachow and Dr. AnkeKohlenberg • Study teams at NIMR-MMRC and KCRI (BarikiMtafya, Christina Manyama, Fred Njeleka, John Joseph, Gibson Kibiki, Davis Kuchaka)